DEAD-box helicase (DDX) family members play differential roles in regulating innate antiviral immune response. However, the physiological roles played by DDX4 in antiviral innate immunity remain unclear. In this study, we unveiled that DDX4 acts as a positive regulatory molecule of Type-I interferon (IFN-I)-mediated antiviral activity. Our findings demonstrate that IFN-I upregulates DDX4 protein levels, and subsequently, overexpression of DDX4 enhances the IFN-I-mediated signaling pathway. This creates a positive feedback loop that amplifies the antiviral response. DDX4 was found to bind with deubiquitinase ubiquitin-specific protease 7 (USP7), leading to the disruption of the interaction between USP7 and suppressor of cytokine signaling 1 (SOCS1) and the subsequent degradation of SOCS1. This process enhances the antiviral function of IFN-I. Our findings provide new insights into the regulatory role of DDX4 in the IFN-I response. IMPORTANCE: DDX4, identified as a putative RNA helicase that modulates RNA secondary structure through RNA binding, is primarily acknowledged for its role in regulating mRNA translation within the germline. Nevertheless, the extent of DDX4’s involvement in the antiviral innate immune response remains largely unexplored. This study presents evidence of a previously unrecognized positive feedback loop between DDX4 and the antiviral response, suggesting that disruption of this loop may serve as a novel mechanism for viral evasion. Furthermore, our findings elucidate a positive regulatory mechanism by which the DDX4/USP7/SOCS1 axis mediates the antiviral activity of Type-I interferon, which provides new insight into strategies for improving the efficacy of IFN-based antiviral therapy.

β transducin repeat-containing protein (β-TrCP) is a Skp1-Cul1-F-box ubiquitin ligase, which plays important roles in controlling numerous signaling pathways. Notably, β-TrCP induces ubiquitination and degradation of inhibitor of NF-κB (IκBα), thus triggering activation of NF-κB signaling. Here, we unexpectedly find that β-TrCP restricts TRAF6-IKK signaling upstream of IκBα induced by lipopolysaccharide (LPS). In LPS-Toll-like receptor 4 (TLR4) pathway, protein kinase D1 (PKD1) is essential for activation of TRAF6-IKK-IκBα signaling including TRAF6 ubiquitination, IKK phosphorylation and subsequent IκBα degradation. We found that LPS promotes binding of β-TrCP to PKD1, and results in downregulation of PKD1 and recovery of IκBα protein level. Knockdown of β-TrCP blocks LPS-induced downregulation of PKD1. Supplement of enough PKD1 in cells inhibits recovery of IκBα protein levels during LPS stimulation. Furthermore, we demonstrate that β-TrCP inhibits LPS-induced TRAF6 ubiquitination and IKK phosphorylation. Taken together, our findings identify β-TrCP as an important negative regulator for upstream signaling of IκBα in LPS pathway, and therefore renew the understanding of the roles of β-TrCP in regulating TLRs inflammatory signaling.

The 2A protease (2A ^pro ) encoded by enterovirus 71 (EV71) serves as an accessory protein with significant involvement in the regulation of EV71 infection and viral replication. EV71-2A ^pro exhibits the ability to suppress various host factors, thereby disrupting the cellular antiviral immune response. However, whether host factors downregulate EV71-2A ^pro remains largely unknown. Here, we discovered that the speckle-type POZ protein (SPOP), functioning as a host E3 ubiquitin ligase, triggers ubiquitination modifications and subsequent degradation of EV71-2A ^pro . SPOP interacts with EV71-2A ^pro and exhibits a dose-dependent downregulation of EV71-2A ^pro levels. Conversely, knockdown of endogenous SPOP upregulated EV71-2A ^pro levels. Subsequent investigations revealed that the SPOP facilitates the lysosome-dependent degradation of EV71-2A ^pro by inducing K48-linked polyubiquitination of EV71-2A ^pro , which ultimately restricts EV71 replication. These findings shed light on the ubiquitination and lysosome-dependent regulation of the crucial EV71-encoded protease, offering a potential therapeutic target for the treatment of EV71 infection. IMPORTANCE: EV71 poses a significant health threat to children aged 5 and below. The process of EV71 infection and replication is predominantly influenced by ubiquitination modifications. Our previous findings indicate that EV71 prompts the activation of host deubiquitinating enzymes, thereby impeding the host interferon signaling pathway as a means of evading the immune response. Nevertheless, the precise mechanisms by which the host employs ubiquitination modifications to hinder EV71 infection remain unclear. The present study demonstrated that the nonstructural protein 2A ^pro , which is encoded by EV71, exhibits ubiquitination and degradation mediated by the host E3 ubiquitin ligase SPOP. In addition, it is the first report, to our knowledge, that SPOP is involved in the host antiviral response.